TMEM41A overexpression correlates with poor prognosis and immune alterations in patients with endometrial carcinoma

Background Expression levels of transmembrane protein 41A (TMEM41A) are related to the progression of malignant tumors. However, the association between TMEM41A expression and endometrial carcinoma (EC) remains unclear. This study aims to identify the roles of TMEM41A expression in the prognosis of patients with EC and its correlation with EC progression. Methods The TMEM41A expression and its correlation with the survival of patients with EC were assessed. Cox regression analysis was used to identify the prognostic factors, while nomograms were used to examine the association between the prognostic factors and the survival of patients with EC. Finally, the link between TMEM41A level and immune microenvironment and RNA modifications was investigated in EC. Results TMEM41A was overexpressed in EC. TMEM41A overexpression could diagnose the EC and evaluate the poor prognosis of patients. Overexpression of TMEM41A was associated with clinical stage, age, weight, histological subtype, tumor grade, and survival status of patients with EC. Clinical stage, age, tumor grade, radiotherapy, and TMEM41A overexpression were factors of poor prognosis in patients with EC. The nomograms revealed the correlation between the TMEM41A level and survival time of patients with EC at 1, 3, and 5 years. Furthermore, TMEM41A overexpression was significantly correlated with the level of the stromal score, immune score, estimate score, NK CD56 bright cells, iDC, NK cells, eosinophils, pDC, T cells, TReg, cytotoxic cells, mast cells, Th17 cells, neutrophils, aDC, NK CD56 dim cells, TFH, Th2 cells, CD8 T cells, macrophages, immune cell markers, and RNA modifications. Conclusions TMEM41A is overexpressed in EC tissues and is associated with the prognosis, immune microenvironment, and RNA modification. Our preliminary studies indicate that overexpression of TMEM41A can potentially serve as a biomarker for EC treatment.


Introduction
Endometrial carcinoma (EC) is a common type of malignant epithelial tumor in women, with 10%-15% of patients with EC being diagnosed at an advanced stage, leading to unfavorable prognosis [1,2]. While surgery and adjuvant treatment can improve the survival time of early patients with EC, there is no effective treatment for those with advanced cancer [1]. Over the years, several genes with crucial biological functions have emerged as research hotspots [3][4][5]. For instance, the combination of PARP inhibitor and PD-1/PD-L1 checkpoint inhibitor could improve the survival time of patients with cancer [3]. Therefore, identifying novel therapeutic targets and early diagnostic markers is critical in cancer.
Recent studies have shown the association between transmembrane protein 41A (TMEM41A) and the occurrences and developments of gastrointestinal tumors [6,7], with significantly higher expression levels of SREBF pathway regulator in golgi 1 (SPRING1) in colorectal cancer than in adjacent normal tissues. In vitro and in vivo studies found that SPRING1 could enhance the growth of colorectal cancer cells, which was related to the increased expression of TMEM41A [6]. TMEM41A was strongly expressed in gastric cancer and linked to lymph node metastasis, distant metastasis, late stage, and poor prognosis. Inhibition of TMEM41A expression could reduce gastric cancer cell migration and metastasis by inhibiting the epithelial-mesenchymal transformation (EMT) process and autophagy [7]. Collectively, these preliminary findings indicate that TMEM41A is crucial in promoting cell growth and migration as an oncogene.
The Cancer Genome Atlas (TCGA) database provides transcriptome data of normal and cancer tissues of patients with cancer [8][9][10], which has been extensively analyzed in various studies [11][12][13]. For example, the down-regulation of B cell translocation gene 1 (BTG1) expression was related to poor prognosis, degree of invasion, and FIGO stage in patients with EC. Overexpression of BTG1 inhibits proliferation, migration, and invasion as well as promotes apoptosis of EC cells, which was related to the EMT process [13]. The correlation between TMEM41A expression and EC remains unclear. Therefore, this study aims to comprehensively analyze the roles and possible mechanisms of TMEM41A in EC and to identify potential candidate molecules for cancer treatment.
tissue samples were paired. The expression levels of TMEM41A in EC were analyzed by expression analysis.

Diagnostic evaluation and prognostic analysis of TMEM41A
Receiver operating characteristic (ROC) analysis on TCGA FPKM data to determine the area under the curve (AUC) and identify its diagnostic value in normal and EC tissues. The data of TMEM41A expression was combined with the prognosis data, and the missing data were excluded. After classifying the samples according to the median expression of TMEM41A, the association between changes in TMEM41A expression and the poor prognosis of patients with EC was identified using survival analysis.

Clinical characteristics analysis
The clinical characteristics of patients with EC were obtained from the TCGA database. These data included the clinical stage, age, weight, histological subtype, tumor grade, and survival status, which were used to group the samples, and TMEM41A expression in these groups was analyzed. Moreover, the median expression of TMEM41A was used to divide the samples into high-and low-expression groups to explore the correlation between TMEM41A levels and clinical characteristics of patients with EC.

Survival analysis in subgroups of patients with cancer
The clinical stage (I, II, III, and IV), weight, height, BMI, and tumor grade of patients with EC were analyzed. The patients were divided into high-and low-expression groups to explore the link between TMEM41A levels and overall survival (OS), disease-specific survival (DSS), and disease progression in patients with EC.

Establishment of TMEM41A-related nomograms based on the Cox method
The clinical data of patients with EC patients and TMEM41A expression in cancer tissues from the TCGA database were matched. The risk indicators of poor prognosis were filtered using the univariate Cox regression analysis with P < 0.05 as the screening criteria. Subsequently, multivariate Cox regression analysis was performed, and its results were used to construct the TMEM41A-related nomograms.

Analysis of immune microenvironment
Single-sample gene set enrichment analysis (ssGSEA) and ESTIMATE algorithms were used to evaluate the levels of immune microenvironment components in EC tissues. Correlation analysis was used to investigate the association between TMEM41A levels and stromal score, dendritic cell (DC), Tcm, immune score, Tgd, B cells, estimate score, Th1 cells, T helper cells, Tem, CD8 T cells, NK CD56dim cells, TFH, Th2 cells, macrophages, cytotoxic cells, Th17 cells, neutrophils, aDC, T cells, TReg, mast cells, eosinophils, pDC, NK cells, iDC, and NK CD56bright cells.

Correlation between TMEM41A expression and immune cell markers
The expression data of immune cell markers and TMEM41A in 554 EC tissues were obtained, and their correlation was investigated using the Spearman correlation analysis.

TIMER database
The TIMER (https://cistrome.shinyapps.io/timer) database was a cancer immune-related database. The relationship between TMEM41A expression and cancer immune cells was analyzed in the gene module of the TIMER database, and the link between TMEM41A expression and cancer immune cell copy number was analyzed in the somatic copy number alteration (SCNA) module of the TIMER database.

Association between the expression levels of TMEM41A and RNA modification
The TMEM41A was input into the RM2Target online database to obtain the TMEM41Arelated RNA modification genes, which were restricted according to the artificial filtering standard of species. The expression data of RNA modification genes and TMEM41A in 554 EC tissues were obtained, and their association was investigated using Spearman correlation analysis.

Statistical analysis
Wilcoxon rank sum test and chi-square test were used to detect the expression levels of TMEM41A in EC. The ROC and survival analyses examined the link between TMEM41A expression and EC diagnosis and patient survival time. Spearman correlation analysis assessed the correlation between TMEM41A expression and immune microenvironment and RNA modification genes. A P-value < 0.05 was considered statistically significant.

Increased expression of TMEM41A is valuable in the diagnosis and prediction of poor prognosis in patients with EC
The expression analysis based on the TCGA data revealed that TMEM41A was overexpressed both in non-paired ( Fig 1A) and paired EC tissues ( Fig 1B). Furthermore, ROC analysis demonstrated that the area under the curve of TMEM41A was 0.667, indicating that TMEM41A had a diagnostic value for EC ( Fig 1C). Finally, survival analysis showed that enhanced TMEM41A expression was associated with shorter OS, DSS, and progression-free interval (PFI) among patients with EC ( Fig 1D-1F).

TMEM41A level correlates with clinicopathological features of patients with EC
Our results highlighted a significant correlation between TMEM41A expression and various clinicopathological features in patients with EC (Fig 2). Specifically, TMEM41A was strongly expressed in cancer tissues from patients with clinical stage II, III, and IV EC compared with those of clinical stage I (Fig 2A-2C). Furthermore, TMEM41A was strongly expressed in cancer tissues of patients above 60 ( Fig 2D) and those with a weight greater than 80 kg ( Fig 2E). Moreover, TMEM41A was strongly expressed in mixed and serous subtype patients, compared with endometrioid subtype. In addition, TMEM41A was strongly expressed in serous patient carcinoma tissues compared to patients with mixed subtypes (Fig 2F-2H). TMEM41A was strongly expressed in cancer tissues of patients with G2 and G3 stages compared with those of G1 stage and in cancer tissues of G3 patients compared with the G2 patients (Fig 2I-2K). Notably, TMEM41A expression levels were significantly enhanced in cancer tissues from deceased patients compared to those from living patients (Fig 2L-2N). Statistical analyses revealed that high-and low-TMEM41A expression was associated with clinical stage, PFI, race, age, DSS, weight, OS, histologic type, histologic grade, and menopausal status in patients with EC (Table 1).

Construction of TMEM41A-related nomograms
Univariate Cox regression analysis revealed that clinical stage, age, histological grade, radiotherapy, and TMEM41A overexpression were risk factors for shorter OS in patients with EC (Table 2). Similarly, clinical stage and TMEM41A overexpression were risk factors for shorter DSS in patients with EC (Table 3). Moreover, clinical stage, histologic grade, and TMEM41A overexpression were risk factors for shorter PFI in patients with EC (Table 4). We further showed the association between adverse factors in patients with EC and patient prognosis to predict patient survival time via nomogram (Figs 6-8).

TMEM41A overexpression associates with the EC immune microenvironment
TMEM41A overexpression was associated with stromal, immune, and estimate scores of cancer samples (Fig 9A-9C). Furthermore, stromal, immune, and estimate scores significantly differed between the high-and low-TMEM41A expression groups (Fig 9D-9F). In EC tissues of the TCGA database, TMEM41A overexpression correlated with the levels of macrophages, CD8 + T cells, TFH, Th2 cells, aDC, cytotoxic cells, eosinophils, iDC, mast cells, neutrophils, NK CD56bright cells, NK CD56dim cells, NK cells, pDC, T cells, Th17 cells, and TReg (Fig 10 and Table 5). The expression levels of 24 immune cell types in the high-and low-TMEM41A expression groups are presented in Fig 11. Additionally, we found that TMEM41A overexpression correlated with tumor purity, CD8 + T cells, macrophage, and neutrophil levels (S1 Fig), and with the copy numbers of CD8 + T cell, neutrophil, and DC in the TIMER database (S2 Fig). Finally, TMEM41A overexpression was also significantly associated with the levels of immune cell markers, including CD8A, CD3D, CD3E, CD2, CSF1R, IL10, IRF5, ITGAM, and CCR7 (Fig 12).  (Table 6).

PLOS ONE
Role of TMEM41A in endometrial carcinoma

Discussion
In recent years, there has been growing interest in identifying new biomarkers for EC. Several studies have focused on identifying and characterizing the roles of novel oncogenes in EC progression and prognosis [14][15][16][17]. For instance, OTU deubiquitinase, ubiquitin aldehyde binding 2 (OTUB2) expression was significantly enhanced in EC, which was associated with poor prognosis of cancer patients. Overexpression of OTUB2 could promote glycolysis and induce proliferation, migration, and invasion of EC cells, while inhibition of the PKM2/PI3K/AKT signaling pathway significantly reversed the oncogenic effects of OTUB2 overexpression on EC cells [15]. Similarly, ubiquitin-specific peptidase 5 (USP5) was significantly upregulated in EC tissues, whereas its inhibition could reduce the migration and proliferation ability of EC cells and induce cell cycle arrest and apoptosis. These effects of USP5 overexpression were associated with excessive activation of the mTOR/4EBP1 signaling pathway [16]. These results suggested that targeting these biomarkers could offer potential therapeutic benefits for patients with EC. TMEM41A is a newly discovered oncogene [6,7]. Studies have shown that the SPRING1 could enhance colorectal cancer cell growth by promoting TMEM41A expression [6]. TMEM41A is also associated with lymph node metastasis, distant metastasis, and poor prognosis in patients with gastric cancer. Inhibition of TMEM41A expression could delay gastric cancer cell metastasis by regulating EMT and autophagy [7]. In this study, we found that TMEM41A was overexpressed in EC and was a potential diagnostic and prognostic marker for the disease. Our comprehensive analysis revealed that TMEM41A overexpression was associated with shorter OS, clinical stage, age, DSS, weight, histological subtype, PFI, tumor grade, race, and menopausal status in patients with EC. Our preliminary findings indicated that TMEM41A played an oncogenic role in EC, which is consistent with previous reports, and indicates that TMEM41A could be a valuable biomarker for predicting EC prognosis and guiding personalized treatment strategies. Nomograms have emerged as an increasingly popular method for predicting the prognosis of patients with cancer [18][19][20]. For example, Wang et al. constructed a nomogram consisting of histological subtype, tumor grade, depth of invasion, cervical involvement, parametrial involvement, and HGB level to accurately predict the risk of lymph node metastasis in patients with EC [19]. Similarly, Li and Yue identified age, race, marital status, FIGO stage, grade, and metastasis as important prognostic factors using univariate Cox regression analysis. The risk factor-related nomogram had predictive power and was associated with the prognosis of patients with EC [20]. In this study, we identified clinical stage, age, histological grade, Immunotherapy has emerged as a promising strategy to improve the prognosis of cancer patients [21][22][23]. For example, patients with esophageal squamous cell carcinoma treated with an anti-programmed death 1 (PD-1) inhibitor plus chemotherapy reported a median OS of 15.3 months and a median progression-free survival (PFS) of 6.9 months, compared with 12.0 months and 5.6 months in the chemotherapy group. This study demonstrated that adding anti-PD-1 therapy to chemotherapy significantly improves survival in patients with advanced or metastatic esophageal squamous cell carcinoma [22]. Therefore, we explored the association between TMEM41A expression and components of the immune microenvironment in EC. We found that TMEM41A overexpression was correlated with EC stromal, immune, and estimate scores and was linked with tumor purity, immune cells (such as the macrophages, CD8 T cells, and TFH), and immune cell markers (such as the CD8A, CD3D, and CD3E). In addition, TMEM41A expression was significantly correlated with the levels of several RNA modification genes, including ALKBH3, ALYREF, FMR1, FTO, HNRNPC, IGF2BP1, IGF2BP3, METTL1, METTL5, PCIF1, RBMX, VIRMA, WTAP, YBX1, YTHDF1, YTHDF2, YTHDF3, YTHDC1, ALKBH5, and HNRNPA2B1. RNA modification genes play important regulatory roles in cancer [24][25][26][27]. For instance, FTO, the m6A modification gene, was highly expressed in metastatic EC tissues, and promoted EC metastasis and invasion by activating the Wnt signaling pathway through the demethylation of HOXB13 mRNA, which eliminated recognition of m6A modification via the YTHDF2 protein [27]. Additionally, the expression level of lncRNA FENDRR was decreased in endometrioid endometrial carcinoma (EEC), while the FENDRR m6A methylation level was significantly increased. YTHDF2 mediated the degradation of FENDRR and promoted cancer cell proliferation by increasing SOX4 expression in EEC [23]. Our findings demonstrated that TMEM41A was associated with immune microenvironment and RNA modification, further suggesting that TMEM41A may play a critical role in EC.
Traditionally, local invasion and histological characteristics have been used as risk factors for the prognosis of patients with EC. However, in recent years, molecular and genomic maps have emerged as promising tools to predict the prognosis of patients with EC, helping in identifying those with low, medium, and high recurrence risks [17,[28][29][30]. Our results revealed  that TMEM41A was upregulated in EC and that its increased expression was related to poor prognosis, immune status, and RNA modification in patients with EC. This study provides new biomarkers for evaluating the prognosis of patients with EC and has the following advantages and disadvantages. We analyzed a large number of tissue samples and prognostic data, ensuring the reliability of our findings. In addition, this study is the first to report the association between TMEM41A expression and EC progression, providing new directions for future   research. However, this study lacked basic research to confirm the current results. Our future studies would focus on collecting cancer tissues and normal adjacent tissues from patients with EC to verify the expression of TMEM41A and explore its impact on the prognosis of patients with EC. Moreover, the effects of inhibiting TMEM41A on the growth and migration of EC cells should be investigated. Additionally, radiomic features are crucial in the screening, diagnosis, and prognosis of patients with EC [30]. Therefore, future studies should combine TMEM41A expression and radiomic features to evaluate the prognosis of patients with EC.

Conclusions
In conclusion, our study demonstrates that TMEM41A exhibits strong expression in EC tissues. TMEM41A could serve as a diagnostic marker and evaluate the poor prognosis of patients with EC. Moreover, the overexpression of TMEM41A is associated with poor prognosis, stromal score, immune score, estimate score, immune cells, cell markers, and RNA modifications. These findings suggest that TMEM41A may serve as a potential biomarker for EC treatment.